SEQUENCING OF DNA ISOLATED FROM COLORECTAL SAMPLE FIXED IN LIQUID FORMALIN : OBSTACLES AND REQUIRED MODIFICATIONS

Abstract

The necessary modifications in the protocol of general purpose DNA
isolation kit to isolate and amplify a target region of genome from colorectal
cancer tissues fixed in liquid formalin were made. It is shown that a one hour
digestion with proteinase K yields enough DNA from formalin fixed colorectal
tissue for subsequent PCR and sequencing. Moreover, using 100% ethanol
instead of standard 50% during DNA binding step in the column improves the
yield. As DNA fragmentation is unavoidable in formalin fixed tissue, PCR
protocol was modified by increasing polymerase concentration to get successful
amplification. Following these modifications, two regions of KRAS and BRAF
genes were amplified and successfully sequenced from three different patients.
These modifications provide a low cost option for Sanger sequencing of DNA
isolated from formalin fixed tissue.